U.S. patent number 4,693,980 [Application Number 06/655,470] was granted by the patent office on 1987-09-15 for new type ii restriction endonuclease mae iii, a process for obtaining it and the use thereof.
This patent grant is currently assigned to Boehringer Mannheim GmbH. Invention is credited to Frank Laue, Rudiger Schmitt, Karl O. Stetter.
United States Patent |
4,693,980 |
Stetter , et al. |
September 15, 1987 |
New type II restriction endonuclease mae III, a process for
obtaining it and the use thereof
Abstract
The present invention provides a restriction endonuclease,
characterized by the palindromic recognition sequence: ##STR1## and
the cleavage position defined by the arrows. The present invention
also provides a process for obtaining this new restriction
endonuclease.
Inventors: |
Stetter; Karl O. (Regensburg,
DE), Schmitt; Rudiger (Niedergebraching/Pentling,
DE), Laue; Frank (Pahl, DE) |
Assignee: |
Boehringer Mannheim GmbH
(Mannheim-Waldhof, DE)
|
Family
ID: |
6225274 |
Appl.
No.: |
06/655,470 |
Filed: |
September 28, 1984 |
Foreign Application Priority Data
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Jan 18, 1984 [DE] |
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3401620 |
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Current U.S.
Class: |
435/199; 435/196;
435/822 |
Current CPC
Class: |
C12N
9/22 (20130101); Y10S 435/822 (20130101) |
Current International
Class: |
C12N
9/22 (20060101); C12N 009/22 (); C12N 009/16 ();
C12N 015/00 (); C12R 001/01 () |
Field of
Search: |
;435/195,196,199,822,91,172.3 |
Other References
Schmid, K. et al., Nuc. Acids Res, vol. 12, No. 6, pp. 2619-2628,
Mar. 1984. .
Balch, W. et al., Micro. Reus, vol. 43, No. 2, pp. 260-264, 285 and
286, 1979..
|
Primary Examiner: Wiseman; Thomas G.
Assistant Examiner: Huleatt; Jayme A.
Attorney, Agent or Firm: Felfe & Lynch
Claims
We claim:
1. A restriction endonuclease capable of recognizing and cleaving a
DNA sequence at a position indicated by the arrows ##STR3##
2. The restriction endonuclease of claim 1 wherein said
endonuclease is characterized by a temperature optimum between
45.degree. and 48.degree. C. and a pH optimum at 8.0/45.degree. C.
in Tris HCl buffer.
3. A process for obtaining the restriction endonuclease of claim 1
comprising the steps of culturing Methanococcus aeolicus DSM 2835
cells and recovering the restriction endonuclease from the
cells.
4. The process of claim 3 comprising recovering the endonuclease
from the cells of Methanococcus aeolicus by digesting the cells to
release an extract therefrom, mixing the extract released from the
digested cells with polyethylenimine up to a concentration of 0.65%
v/v, separating off insolubles and leaving a supernatant, mixing
the supernatant with ammonium sulphate in an amount of up to 60 to
95% saturation to form a precipitated fraction and recovering the
precipitated fraction.
5. The process of claim 4, further comprising purifying the
ammonium sulphate precipitated fraction by at least one process
selected from the group consisting of molecular sieve
fractionation, chromatography over a weakly basic anion exchanger,
chromatography over a weakly acidic cation exchanger, and affinity
chromatography.
6. The process of claim 5, wherein carrier-fixed heparin is used
for the affinity chromatography.
Description
The present invention is concerned with a new Type II restriction
endonuclease MaeIII, with a process for obtaining it and with the
use thereof.
Type II restriction endonucleases are endodeoxyribonucleases which
are able to recognize and cleave certain DNA at nucleotide
sequences. Phosphodiester bridges are thereby hydrolysed in the
target sequence, namely one in each polynucleotide chain.
Therefore, Type II restriction endonucleases are valuable for the
analysis of DNA molecules.
Specific Type II restriction endonucleases are admittedly already
known for numerous recognition sequences, but there is still a need
for the provision of further Type II restriction endonucleases
which are specific for such recognition sequences for which
restriction endonucleases have not been recognized.
Therefore, it is an object of the present invention to provide a
new restriction endonuclease which is able specifically to
recognize and cleave a sequence which hitherto have not been
recognized by any such enzyme.
Thus, according to the present invention, there is provided a
restriction endonuclease which is characterized by the palindromic
recognition sequence: ##STR2## and the cleavage position defined by
the arrows.
The new Type II restriction endonuclease according to the present
invention, which in the following is called MaeIII, has a
temperature optimum of 45 to 48.degree. C. and a pH optimum at
8.0/45.degree. C. in Tris/HCl buffer. Further optimum reaction
parameters are 350 mM NaCl, 10 to 18 mM Mg.sup.2+, 0 to 20 mM
2-mercaptoethanol. The presence of magnesium ions is essential for
the activity of the enzyme.
As mentioned above, the enzyme acts upon palindromic structures and
thus recognizes a self-complementary structure in which the
complementary strand of the DNA has the identical sequence in the
opposite-running direction.
The recognition sequence and the point of cleavage of the enzyme
can be ascertained as follows: the plasmid pBR322 is completely
digested with HinfI. The HinfI fragments B and C (517 bp and 506
bp, respectively) are isolated, their 3'-ends are marked with
alpha-[.sup.32 P] dATP and Klenow polymerase and subsequently
cleaved with AluI. From the marked fragments which hereby results,
there is isolated and sequenced the 330 bp fragment (pBR322,
position 3037 to 3366, length of the fragment including single
strand ends).
An aliquot of the 330 bp fragment is cleaved with the enzyme
according to the present invention resulting in two fragments. The
cleavage position 3293 lying close to the 3'labeled end was
determined.
The length of the HinfI/MaeIII fragment is determined in sequence
gels. The HinfI/MaeIII fragment thereby runs in the gel like the
"A" in the sequence ladder which stands 5'-adjacent to the
recognition sequence 5'-GTNAC-3'. Therefore, it terminates with the
nucleotide G of the recognition sequence. The cleavage point of
MaeIII is thus 5'-positioned to the 5'-flanking nucleotide G.
The new endonuclease MaeIII is obtained, according to the present
invention, by culturing Methanococcus aeolicus DSM 2835 and
recovering the enzyme from the cells. For the recovery, there can
be used conventional biochemical purification methods, whereby, in
each of the fractions obtained, the presence of the enzyme can be
demonstrated on the basis of the cleavage of its recognition
sequence. As substrate, there can be used for example, pBR322-DNA.
The DNA fragments obtained are separated electrophoretically in
agarose gels in the buffer systems conventional for fragment
separation in the presence of ethidium bromide.
The microorganism used for obtaining the enzyme grows anaerobically
in Medium III (Microbiol. Reviews, 43, 260-296/1979) on H.sub.2
/CO.sub.2 or on formate. It forms regular to irregular cocci of
about 2 .mu.m diameter, individually and in pairs. On agar, there
are formed round, convex, pale ochre-coloured colonies of about 2
mm diameter. The microorganism is gram-negative. The cell
integument consists of protein subunits. Growth takes place at a
temperature of from 25.degree. to 50.degree. C., the temperature
optimum being 45.degree. C. (2 hours duplication time). Growth
takes place in the presence of 1.5 to 5% and optimally of 4% sodium
chloride. The DNA base composition is about 28.6% G+C. Therefore,
the microorganism differs from the known Methanococci by a somewhat
lower GC content of the DNA, by the optimal growth temperature of
45.degree. C., by the markedly larger cells and by the presence of
new restriction enzymes. Methanococcus aeolicus has been deposited
at the Deutsches Sammlung von Mikroorganismen, Gesellschaft fur
Biotechnologische Forschung GmbH, Grisebachstrasse 8, 3400
Gottingen, Federal Republic of Germany, and bears Accession Number
DSM 2835.
In a preferred embodiment of the process according to the present
invention, the cells are digested, the extract is mixed with
polyethyleneimine up to a concentration of 0.65%, the precipitate
is separated off and from the supernatant there is obtained the
fraction precipitating out between 60 and 95% ammonium sulphate
saturation.
For the digestion, there can be used the conventional mechanical
and chemical methods, for example high pressure dispersion,
ultrasonics or enzymatic digestion.
Further purification of the ammonium sulphate fraction containing
the new enzyme is preferably conducted by molecular sieve
fractionation, chromatography over anion exchangers and over cation
exchangers, as well as final affinity chromatography. As molecular
sieve material, there has proved to be useful the product which is
commercially available under the designation Ultrogel AcA 34, this
being an acrylamide/agarose heteropolymer of 3% acrylamide and 4%
agarose. As anion exchangers, there can be used carrier materials
based on sepharose, cellulose or synthetic polymers which are
modified with diethylaminoethyl substituents, for example the
products of Pharmacia, Uppsala, Sweden, which are commercially
available under the designation DEAE-Sephacel.
As cation exchangers, there are preferably used phosphate
group-containing substances, preferably carbohydrates, for example
cellulose phosphate and the like. For the affinity chromatography,
carrier-fixed heparin, for example heparin-sepharose, has proved to
be especially useful.
The following examples are given for the purpose of illustrating
the present invention:
EXAMPLE 1
Methanococcus aeolicus DSM 2835 is allowed to grow anaerobically at
45.degree. C. for 3 days in minimal formate medium, which is
described in detail hereinafter, whereafter it is harvested in the
stationary phase. 35 g of the cell paste so obtained are suspended
in 70 ml digestion buffer (40 mM Tris/HCl, pH 8.0/4.degree. C.; 0.1
mM EDTA (ethylenediamine-tetraacetic acid); 7 mM 2-mercaptoethanol
and 0.2 mM PMSF (phenylmethanesulphonyl fluoride). The cells are
then digested twice by high pressure dispersion in a precooled
pressure cell at 1100 bar=16,000 PSI.
To the digestion suspension ammonium chloride is added to a final
concentration of 0.3M. Subsequently, 7 ml of a 10%
polyethyleneimine solution are added to give a final concentration
of 0.65% (v/v). After leaving to stand for 30 minutes at 4.degree.
C., the precipitate formed is centrifuged off for 60 minutes at
27,300 g or 23,000 g and discarded. The supernatant is mixed with
solid ammonium sulphate up to 60% saturation, left to stand for 2
hours at 4.degree. C. and then centrifuged off for 60 minutes at
27,300 g or 23,000 g. The resultant supernatant is again brought to
90% saturation with sold ammonium sulphate. After 16 hours at
4.degree. C., the ammonium sulphate precipitate is centrifuged off
for 60 minutes at 27,300 g and further worked up.
The minimal medium referred to above has the following
composition:
______________________________________ dissolve: g/liter
______________________________________ KCl 0.32 g
MgCl.sub.2.6H.sub.2 O 2.75 g MgSO.sub.4.7H.sub.2 O 3.45 g NH.sub.4
Cl 0.25 g CaCl.sub.2.2H.sub.2 O 0.15 g K.sub.2 HPO.sub.4 0.15 g
NaCl 18 g mineral elixir (see below) 10 ml Fe(NH.sub.4).sub.2
(SO.sub.4).sub.2.7H.sub.2 O 2 mg NaHCO.sub.3 (added at end) 5.5 g
resazurin 0.1% 1 ml sodium formate 15 g sodium tungstate 3.3 mg
______________________________________
add 50 ml of reducing agent, consisting of 12.5 g/liter sodium
sulphide, while allowing nitrogen to bubble through, 75 ml freshly
prepared 1N sodium hydroxide solution and 1 ml 0.1% resazurin,
adjust the pH value with formic acid to 6.9, make up to 1 liter and
allow nitrogen to bubble through.
The mineral elixir referred to above has the following
composition:
______________________________________ g/liter
______________________________________ Titriplex I 1.5 g
MgSO.sub.4.7H.sub.2 O 3.0 g MnSO.sub.4.2H.sub.2 O 0.5 g NaCl 1.0 g
FeSO.sub.4.7H.sub.2 O 0.1 g CoSO.sub.4 or CoCl.sub.2 0.1 g
CaCl.sub.2.2H.sub.2 O 0.1 g ZnSO.sub.4 0.1 g CuSO.sub.4.5H.sub.2 O
0.01 g KAl(SO.sub.4).sub.2 0.01 g H.sub.3 BO.sub.3 0.01 g Na.sub.2
MoO.sub.4.2H.sub.2 O 0.01 g slowly adjust pH value to 6.5 with 5 N
KOH ______________________________________
EXAMPLE 2
The ammonium sulphate precipitate obtained according to example 1
is taken up with TEMG buffer (40 mM Tris/HCl, pH 8.0/4.degree. C.;
0.1 mM EDTA; 7 mM 2-mercaptoethanol; 10% v/v glycerol) and 0.5M
sodium chloride solution and applied to an Ultrogel AcA-34
molecular sieve column of 3.times.100 cm. This column is eluted
with TEMG buffer +0.5M sodium chloride solution and the eluate
fractions with MaeIII activity are combined.
The combined eluate fractions are chromatographed on an anion
exchanger column (DEAE-Sephacel; 2.times.10 cm) equilibrated with
TEMG buffer. After washing with 2 column volumes of TEMG buffer,
the enzyme is eluted with a linear gradient of 0 to 1M sodium
chloride in TEMG. The enzyme appears in the fractions with 0.1 to
0.15M sodium chloride. The active fractions are combined and
dialysed against TEMG buffer. The dialysate is chromatographed on a
cation exchanger column (cellulose phosphate P 11; 1.times.10 cm)
equilibrated with TEMG buffer. Washing and elution take place as in
the case of the anion exchanger column. MaeIII is eluted between
0.55 and 0.65M sodium chloride. The combined enzyme-containing
fractions are again dialysed against TEMG buffer and the dialysate
chromatographed over an affinity chromatography column
(heparin-sepharose CL-6B; 1.times.5 cm) equilibrated with TEMG
buffer. Washing and elution again takes place as described in the
case of the anion exchanger column. MaeIII is eluted between 0.65
and 0.85M sodium chloride. The active fractions are combined and
dialysed against 20 mM Tris/HCl buffer, pH 8.0, 4.degree. C.,
containing 0.1 mM EDTA, 10 mM 2-mercaptoethanol, 100 mM sodium
chloride, 50 vol.% glycerol, 0.01 vol.% Triton X100, and stored at
-20.degree. C. The activity is about 100 U MaeIII (definition of
activity: 1 U=1 .mu.g pBR322-DNA/hour at 45.degree. C. completely
cleaved).
Determination of activity
Into a mixture of 5 .mu.l incubation buffer, containing 0.03M
Tris/HCl, pH 8.0/45.degree. C., 1.75M sodium chloride, 0.07M
magnesium chloride, 0.035M 2-mercaptoethanol and 0.05 vol.% Triton
X100, there are introduced 14 .mu.l water and 5 .mu.l pBR322-DNA (4
OD/ml), as well as 1 .mu.l MaeIII solution (1 U/.mu.l). The
solution is maintained at 45.degree. C. for 1 hour, cooled on ice
and mixed with 5 .mu.l cold stop solution, containing 7M urea, 20%
w/v sucrose, 0.06M EDTA and 0.01% w/v bromophenol blue. It is then
separated electrophoretically on 1% agarose gel for 3 to 4 hours at
100 V. The bands obtained are identified in comparison with
suitable DNA length standards.
* * * * *